经静脉途径纳米氧化锆颗粒对大鼠脾脏的毒性研究

发布时间：2018-04-21 05:46

本文选题：氧化锆 + 纳米颗粒　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景纳米材料是由纳米颗粒所构成的材料。纳米颗粒的定义是单个颗粒的三维尺寸中至少有一个维度的尺寸在1～100 nm的材料。由于颗粒直径小,纳米材料具有了大块固体材料所不具备的多种理化特性,也因此被广泛用于工业生产的各个领域。但纳米材料本身并非完全无害的。随着纳米材料应用范围的扩展,相关领域的学者也开始关注其生物安全性问题。大量的实验研究显示纳米材料在研发、生产以及应用的过程中,纳米颗粒可进入水体、土壤等自然环境,再通过多种途径进入人体,经血液循环进入组织器官和细胞中,进而产生一系列的损伤效应[1]。纳米氧化锆材料在口腔医学领域也有广阔的应用空间。通过使用纳米氧化锆对传统口腔材料进行改性,可极大的改善传统材料的性能[2-4]。但作为纳米材料,纳米氧化锆的生物安全性问题同样值得关注。然而,现有文献关于纳米氧化锆的毒性研究十分有限。本研究拟建立纳米氧化锆颗粒的体内染毒模型,通过电感耦合等离子体质谱技术,分析纳米氧化锆颗粒在心、脾、肺、肾中的分布及代谢规律;通过血清中炎症因子指标的变化分析颗粒对脾脏的影响;通过组织病理技术、生物化学技术进一步分析其对脾脏的毒性作用,为纳米氧化锆的毒性研究提供新思路。研究目的:(1)建立纳米氧化锆颗粒急性染毒模型,观察颗粒在大鼠体内的分布情况。(2)通过血清中炎症因子指标的变化分析纳米氧化锆对脾脏的影响。(3)研究纳米氧化锆对大鼠脾组织的损伤作用。(4)探究纳米氧化锆对脾脏毒性作用的机制。材料与方法:第一部分:纳米氧化锆颗粒的表征(1)透射电子显微镜检测纳米原始粒径及形态。(2)电位测定仪检测纳米氧化锆颗粒在混悬液中的电位值及水合粒径。(3)能谱分析仪检测纳米颗粒的化学元素构成。(4)氮气吸附实验检测纳米颗粒的比表面积。第二部分:锆元素的体内分布及血清中炎症因子含量的分析(1)构建动物模型模型构建前选用0.9%的无菌生理盐水将纳米氧化锆颗粒分散均匀并配置颗粒混悬液,然后对6周龄的雄性wistar大鼠进行混悬液的单次尾静脉注射处理(20 mg/kg b.w.)。(2)锆元素的生物学分布在给药后的第1 d、7 d、14 d,分别收集对照组和实验组大鼠的心、脾、肺、肾组织,经浓硝酸与H202消解后,采用电感耦合等离子体质谱分析检测各样品中锆元素含量。(3)血清中炎症因子含量的分析在给药后的第1d、7d、14d,分别收集对照组和实验组大鼠的血清,参照液相蛋白分析试剂盒说明准备样品及标准品,采用液相蛋白分析仪测定血清中炎症因子的含量。第三部分:纳米氧化锆对脾组织的损伤研究(1)脾组织的病理学观察收集对照组7d与实验组7d、14d大鼠的新鲜脾组织(n=3),浸泡于4%的中性甲醛溶液完全固定后,制作石蜡切片。采用常规的苏木精伊红染色观察脾组织的结构变化。(2)脾组织的免疫组织化学染色观察进行常规苏木素伊红染色的同时,分别采用Ki-67与末端标记法凋亡染色技术观察脾组织的增殖与凋亡情况。第四部分:纳米氧化锆毒性作用的机制研究(1)组织匀浆的制备与总蛋白含量的测定分别收集实验组(1d、7 d、14 d)和对照组(1d、7 d、14 d)大鼠的脾组织,用组织匀浆机制备组织匀浆,离心收集后稀释至所需浓度。根据总蛋白定量试剂盒的说明,检测匀浆中的蛋白含量。(2)氧化应激相关酶活性的检测同样使用实验组(1d、7 d、14 d)和对照组(1d、7 d、14 d)大鼠脾组织所制备的匀浆。按超氧化物歧化酶试剂盒之操作说明完成检测样品制备。采用荧光酶标仪测定样品OD值,并计算出组织中超氧化物歧化酶的活性。(3)脂质过氧化产物的含量检测使用适当浓度的组织匀浆,按照丙二醛试剂盒之操作说明制备检测样品。样品经水浴加热后反应完全,测定样品OD值,并计算丙二醛含量。结果:第一部分:透射电子显微镜观察结果显示纳米氧化锆颗粒近似球形,原始粒径平均约为38 nm。电位测定仪显示纳米颗粒在水溶液中的电动电位为30.6 mV,水合粒径约为181.2 nm。能谱仪结果示纳米颗粒的元素组成符合氧化锆的特点,无其他杂质。氮气吸附实验结果示纳米氧化锆颗粒的比表面积为33.06 m2/g。第二部分:生物学分布检测结果显示,锆元素在对照组大鼠的心、脾、肺、肾组织中未检出,在实验组大鼠的心、肾组织中未检出,实验组大鼠脾、肺组织中的含量显著高于对照组。分析实验组脾、肺组织中锆元素的含量变化,脾组织中锆元素的含量14 d7d1d,提示锆元素含量随时间累积而逐渐升高。肺组织中锆元素的含量7 d1 d=14 d,锆元素含量先升后降。血清中IL-1α的含量,实验组7 d显著高于对照组(1d、7 d、14 d)与实验组(1d、14 d);血清中IL-1β含量,实验组1d显著高于对照组(1d、7 d、14 d)与实验组(7d、14d);血清中IL-2的含量各组无统计学差异;血清中IL-6的含量各组无统计学差异;血清中IL-12含量,实验组1 d显著低于对照组(1 d、7 d、14 d)与实验组(7 d、14 d);实验组7 d显著高于对照组(1d、7 d、14 d)与实验组(1d、14 d);对照组(1d、7 d、14 d)与实验组14 d间无统计学差异;血清中TNF-α的含量,实验组7 d显著高于对照组(1d、7 d、14 d)与实验组(1d、14d);实验组14d低于对照组(1d、7d、14d)与实验组(7d);对照组(1d、7 d、14 d)与实验组1d间无统计学差异。第三部分:苏木素伊红染色结果显示,实验组7 d、14 d动物脾组织相较对照组7 d无明显组织病理学改变,对照组7 d、实验组(7 d、14 d)的脾组织切片中均清晰可见白髓、红髓及脾小梁等正常结构。Ki-67染色结果,实验组7 d脾组织中细胞阳性率与对照组7 d无明显差异,但对照组7 d脾组织的红髓与白髓中可见阳性细胞分布规律,而实验组7 d的脾组织中阳性细胞主要集中于白髓中,红髓中少见阳性细胞分布,实验组14 d的细胞阳性率低于对照组7 d和实验组7 d。末端标记法凋亡染色结果示,对照组7 d脾组织中可见少量阳性胞,实验组7 d脾组织中的部分区域阳性细胞数多于对照组7 d,实验组14 d的阳性细胞比率与对照组7 d无差异。提示实验组7 d脾组织中部分区域凋亡细胞数目增加。第四部分:氧化应激相关酶活性检测结果显示,实验组1d、14 d的脾组织中超氧化物歧化酶活力较对照组显著下降(P0.01)。表明给药后实验组动物脾组织中该酶的活性受到了损害;脂质过氧化产物的含量检测表明,丙二醛的含量在给药后的实验组动物中脾组织中未有明显改变。结论:1.纳米氧化锆颗粒可沉积于脾、肺组织中,并引起脾组织炎症反应和氧化应激。2.纳米氧化锆颗粒沉积可引起脾红髓细胞增殖减少、诱导脾组织细胞凋亡,引起功能损伤。3.纳米氧化锆颗粒在脾脏中随时间而逐渐沉积,并至少可维持14 d。4.纳米氧化锆颗粒对脾脏损伤的机制与氧化应激密切相关。
[Abstract]:Background nanomaterials are made of nanoparticles. The nanoparticles are defined as a material with at least one dimension in the dimension of a single particle in a dimension of 1~100 nm. Due to the small diameter of the particles, the nanomaterials have many physical and chemical properties that the bulk solid materials do not have, and are widely used in industrial production. Nanomaterials themselves are not completely harmless. With the expansion of the application scope of nanomaterials, scholars in the related fields have also begun to pay attention to their biological safety problems. A large number of experimental studies show that nanomaterials can enter the water, soil and other natural environments in the process of development, production and application of nanomaterials. A variety of ways to enter the human body and circulate through the blood into tissues and organs and cells, and then produce a series of damage effects [1]. nano zirconia materials have wide application space in the field of Stomatology. By using nano zirconium oxide to modify traditional oral materials, it can greatly improve the performance of traditional materials [2-4]. but as a Na The biosafety of rice materials and nano zirconia is also worthy of concern. However, the current literature about the toxicity of nano zirconia is very limited. This study is to establish an in vivo toxicity model of nano zirconia particles. The distribution of nano zirconia particles in the heart, spleen, lung and kidney is analyzed by inductively coupled plasma mass spectrometry. And the regulation of metabolism; the effect of granule on spleen was analyzed through the changes of inflammatory factors in serum. Through histopathology, biochemical technology was used to further analyze its toxic effect on the spleen and provide new ideas for the study of the toxicity of nano zirconia. (1) to establish an acute poisoning model of nano zirconia particles and observe the particles. Distribution in rats. (2) analysis of the effect of nano zirconia on spleen by changes of inflammatory factors in serum. (3) study the damage effect of nano zirconia on spleen tissue of rats. (4) explore the mechanism of the toxicity of nano zirconia to spleen. Material and method: the first part: characterization of nano zirconia particles (1) transmission The original particle size and morphology of nanoscale nanoparticles were detected by electron microscopy. (2) potential measurement instrument was used to detect the potential value and hydrous particle size of nano zirconia particles in suspension. (3) the chemical element composition of nanoparticles was detected by the energy spectrum analyzer. (4) the specific surface area of nano particles was detected by nitrogen adsorption experiment. The second part: in vivo distribution of zirconium elements and in serum Analysis of the content of inflammatory factors (1) before the construction of animal model model, 0.9% of the aseptic saline was selected to disperse the nano zirconia particles evenly and to configure the granular suspension. Then, the male Wistar rats of 6 weeks old were treated with a single tail vein injection (20 mg/kg b.w.). (2) the biological distribution of zirconium elements was given after the drug delivery. First D, 7 d, 14 d, the heart, spleen, lung and kidney tissue of the control group and the experimental group were collected respectively. The content of zirconium elements in the samples was detected by inductively coupled plasma mass spectrometry after the digestion of concentrated nitric acid and H202. (3) the serum levels of inflammatory factors were analyzed at 1D, 7d, and 14d after administration, and the blood of the rats in the control group and the experimental group were collected respectively. The content of inflammatory factors in serum was determined by liquid phase protein analyzer. The third part: Study on the damage of the spleen tissue by nano zirconium oxide (1) the pathological observation of spleen tissue (1) the control group 7d and experimental group 7d, the fresh splenic tissue of 14d rats (n=3) were soaked in 4%. After the sex Formaldehyde Solution was completely fixed, the paraffin section was made. The structural changes of spleen tissue were observed with routine hematoxylin eosin staining. (2) immuno histochemical staining of spleen tissue was used to observe the routine hematoxylin staining of routine hematoxylin, and the proliferation and apoptosis of spleen tissue were observed by Ki-67 and terminal labeling method. The fourth part: Study on the mechanism of the toxicity of nano zirconia (1) the preparation of tissue homogenate and the determination of the total protein content of the experimental group (1D, 7 d, 14 d) and the control group (1D, 7 d, 14 d) of the spleen tissue of the rats, the tissue homogenate was prepared by the homogenate mechanism, and then diluted to the required concentration after centrifugation. According to the description of the total protein quantitative reagent box, Detection of protein content in homogenate. (2) detection of oxidative stress related enzyme activity was also used in the homogenate prepared by the experimental group (1D, 7 d, 14 d) and the control group (1D, 7 d, 14 d). The sample was prepared by the operation instructions of the superoxide dismutase Kit. Activity of oxide dismutase. (3) the content of lipid peroxidation products was detected by proper concentration of tissue homogenate, and the test samples were prepared according to the operation instructions of the malondialdehyde kit. The sample was heated by water bath and reacted completely, measured the sample o value and calculated the content of malondialdehyde. Results: the first part: transmission electron microscope observation results show that The nano zirconia particles are approximately spherical. The average size of the original particle size is about 38 nm. potential meter. The electrokinetic potential of nano particles in aqueous solution is 30.6 mV, and the hydrous particle size is about 181.2 nm.. The results show that the element composition of the nanoparticles conforms to the characteristics of zirconia and has no other impurities. The experimental results of nitrogen adsorption show nano zirconia particles. The specific surface area was 33.06 m2/g. second parts: the results of biological distribution detection showed that the zirconium element was not detected in the heart, spleen, lung and kidney tissue of the control group, and was not detected in the heart and kidney tissue of the experimental group. The content of the spleen and lung tissue in the experimental group was significantly higher than that in the control group. The content of zirconium elements in the spleen was 14 d7d1d, suggesting that the content of zirconium elements increased gradually with time. The content of zirconium elements in the lung tissue was 7 D1 d=14 D, the content of zirconium elements increased first and then decreased. The content of IL-1 alpha in the serum, 7 d in the experimental group was significantly higher than that of the control group (1D, 7 d, 14 d) and the experimental group (1D, 14 d), and the content of IL-1 beta in the serum. Compared with the control group (1D, 7 d, 14 d) and the experimental group (7d, 14d), there was no statistical difference in serum IL-2 content, and there was no statistical difference in the content of IL-6 in serum. The content of IL-12 in the serum was significantly lower than that of the control group (1 D, 7 d, 14 d) and the experimental group (7, 14), and the experimental group 7 was significantly higher than that of the control group (7, 14 concerned) and experimental group. 14 d); there was no statistical difference between the control group (1D, 7 d, 14 d) and the experimental group 14 d; the content of TNF- alpha in the serum and the experimental group 7 d were significantly higher than that of the control group (1D, 7 d, 14 d) and the experimental group (1D, 14d); the experimental group was lower than the control group, and the control group was not statistically different from the experimental group. Third: Su Mu Su Hong The staining results showed that the experimental group 7 d, 14 d animal spleen tissue compared with the control group 7 d no obvious histopathological changes, 7 d in the control group, the experimental group (7 d, 14 d) of the spleen tissue sections clearly visible white pulp, red pulp and Pi Xiaoliang and other normal structure.Ki-67 staining results, the experimental group 7 d spleen tissue positive rate and the control group 7 d no significant difference, But the distribution of positive cells in the red pulp and white pulp of the spleen tissue of the control group was 7 d, while the positive cells in the spleen tissue of the experimental group were mainly concentrated in the white pulp and the rare positive cells in the red pulp were mainly concentrated in the 7 d of the experimental group. The positive rate of the 14 d in the experimental group was lower than that of the control group 7 d and the experimental group 7 d. terminal labeling method apoptosis staining results, and the control group 7 d spleen group. The number of positive cells in the 7 d splenic tissues in the experimental group was more than 7 d in the control group. The ratio of positive cells to 14 d in the experimental group was not different from that of the control group 7 d. It suggested that the number of apoptotic cells in the part of the spleen tissue of the experimental group increased. Fourth: the test results of the oxidative stress related enzyme activity showed that the experimental group was 1D, 1, 1. The activity of superoxide dismutase in the spleen tissue of 4 D was significantly lower than that in the control group (P0.01). It showed that the activity of the enzyme in the spleen tissues of the experimental group was damaged after the administration, and the content of lipid peroxidation products showed that the content of malondialdehyde was not significantly changed in the spleen tissue of the experimental group after the administration. Conclusion: 1. nano zirconia. The particles can be deposited in the spleen, lung tissue, and the inflammatory response of the spleen tissue and the oxidative stress of the spleen.2. nanoparticles can cause the proliferation and decrease of the splenic red marrow cells and induce the apoptosis of the spleen tissue cells. The.3. nano zirconia particles are gradually deposited in the spleen with time, and at least 14 d.4. nanoscale zirconia nanoparticles can be maintained. The mechanism of granule damage to spleen is closely related to oxidative stress.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1;TB383.1

【参考文献】